DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a new paradigm to decipher DNA sequences. In the grant application, we will pursue the development of a DNA sequencing system that is a hybrid between Sanger dideoxy chain terminating reaction and SBS using molecular engineering approaches. In this approach, four nucleotides, modified as reversible terminators (RTs, 3'-O-R1-dNTPs) by capping the 3'-OH with small reversible moiety (-R1) so that they are still recognized by DNA polymerase as substrates, are used in combination with a small amount of four cleavable fluorophore labeled dideoxynucleotide permanent terminators (PTs, ddNTPs-R2-fluorophore) to perform SBS on a DNA chip. DNA sequences will be determined by the unique fluorescence emission of each fluorophore on the ddNTPs. Upon removing the 3'-OH capping group on the RTs and the fluorophore from the PTs, the polymerase reaction will reinitiate and the DNA sequence can be continuously determined. We have recently demonstrated the feasibility of generating good quality sequencing data using this method. We will further develop this new method so that it can be readily used in the new generation of DNA sequencing by synthesis systems based on fluorescence detection. In addition, we will develop a walking strategy for SBS to use the immobilized DNA templates multiple times to increase the readlength of the SBS. We anticipate that up to 100 bp of continuous sequences will be produced by this approach, which can be used to pursue a variety of biomedical research projects.